JPWO2011111824A1 - Method for proliferating cardiomyocytes using microRNA - Google Patents

Abstract

An object of the present invention is to provide a method for proliferating cardiomyocytes using miRNA that promotes the proliferation of cardiomyocytes, a vector for treating heart disease, a pharmaceutical composition for treating heart disease, and the like. A method for proliferating cardiomyocytes using miRNA having an effect of promoting proliferation of cardiomyocytes, a vector for treating heart disease containing the miRNA, a pharmaceutical composition for treating heart disease containing the vector, and the like are provided. The miRNA is preferably a miRNA selected from the group consisting of mature miRNAs miR-148a, miR-148b, miR-152 and miR-373, precursors of the miRNA, and variants and similar substances thereof. .

Description

  The present invention relates to a method for growing cardiomyocytes using microRNA, a vector for treating heart disease, and a pharmaceutical composition for treating heart disease.

  Since the cardiomyocytes in adults have lost cell division ability, they are not replenished once they are necrotized or lost by exposure to various stresses such as ischemia and myocarditis. As a result, the remaining cardiomyocytes try to maintain cardiac function by compensatory hypertrophy, but if that state persists and exceeds the permissible range of myocardial tissue, further myocardial cell fatigue and death occurs. Occurs and eventually exhibits decreased myocardial function, i.e. heart failure.

  Heart diseases such as heart failure are the second leading cause of death in Japan. In addition, the prognosis of patients with heart disease is extremely poor, and the 5-year survival rate is only about 50%. Therefore, the development of an effective heart failure treatment method is considered to be extremely beneficial from the viewpoint of medical welfare and medical economy.

  Until now, cardiotonic agents such as digitalis and xanthine preparations that increase myocardial contractility have been used as therapeutic agents for heart failure. However, it is known that long-term administration of these drugs may worsen the disease state. ing. Recently, treatments with drugs that relieve excessive cardiac load due to enhancement of the sympathetic nervous system and renin-angiotensin system, such as β-blockers and ACE inhibitors, have become mainstream, but these are symptomatic treatments. However, it does not restore the damaged heart tissue itself.

  On the other hand, heart transplantation is a fundamental treatment for severe heart failure, but this method is a general treatment because of problems such as lack of organ donors, medical ethics, and the physical or economic burden on patients. Is difficult to use.

  MicroRNA (hereinafter referred to as “miRNA”) is non-coding RNA having a length of 21 to 25 bases present in cells. It has been widely conserved from nematodes to mammals, and has recently attracted attention as a molecule that plays an important role in regulating the expression of various genes in vivo.

  miRNA is first transcribed as a miRNA primary transcript (pri-miRNA) of about several hundred to several thousand bases from a miRNA gene present on genomic DNA. Next, it is processed by a RNase III enzyme called Drosha in the nucleus to become a precursor miRNA (pre-miRNA) having a hairpin structure of about 70 bases. It is then transported from the nucleus to the cytoplasm by Exportin-5 and processed by an RNase III enzyme called Dicer to form a double-stranded mature miRNA (mature miRNA) of 21-25 bases. In mature miRNA, one strand of the miRNA forms a complex with a protein called RNA-induced repression complex (RISC), binds to the mRNA of the target gene having a complementary sequence, and suppresses the expression of the target gene. It is known (Non-patent Documents 1 and 2).

  It is estimated that there are about 1000 miRNAs per species, each of which is said to regulate the expression of multiple target genes and control about 1/3 of the entire protein-encoding gene. (Non-Patent Document 3). In addition, it has become clear that miRNAs are involved in development, differentiation, biological reactions during viral infection, and the like, and it is suggested that they are associated with human diseases. In particular, regarding the relationship between cancer and miRNA, there are many miRNAs with different expression patterns in normal and cancer tissues, which strongly suggests the involvement of miRNA in the carcinogenesis process, miRNAs that promote carcinogenesis, and tumor suppression The presence of both miRNAs has been reported (Non-Patent Documents 4, 5, and 6).

  As for heart disease, miRNA expression profile analysis in normal and diseased hearts has been reported by many research groups. Some of them have also been analyzed for specific functions.For example, miR-195 expression is increased in cardiac hypertrophy, and in the transgenic mice in which the miRNA is forcibly expressed, the heart is enlarged and dilated myocardium. MiR-1, a miRNA whose expression is decreased due to cardiac hypertrophy, suppresses cardiomyocyte proliferation (Patent Document 2, Non-patent Document) Reference 8) has been reported.

WO2009 / 062169 WO2006 / 107826

He & Hannon, Nature Reviews Genetics, 5: 522-531, (2004) Bartel, Cell, 116: 281-297, (2004) Berezikov et al., Cell, 120: 21-24, (2005) Esquela-Kerscher & Slack, Nature Reviews Cancer, 6: 259-269, (2006) Kent & Mendell, Oncogene, 25: 6188-6196, (2006) Zhang et al., Developmental Biology, 302: 1-12, (2007) van Rooij et al., Proceedings of the National Academy of Sciences of the United States of America, 103: 18255-18260, (2006) Zhao et al., Cell, 129: 303-317, (2007)

  An object of the present invention is to identify a miRNA that promotes the proliferation of cardiomyocytes, and to provide a method for growing cardiomyocytes using the miRNA, a vector for treating heart disease, a pharmaceutical composition for treating heart disease, and the like.

  In order to solve the above-mentioned problems, the present inventors have conducted research focusing on microRNA (hereinafter referred to as “miRNA”) as a factor that regulates the proliferation of cardiomyocytes, and analyzed the miRNA that regulates the proliferation of cardiomyocytes. Went. As a result of intensive studies, the present inventors have succeeded in identifying a plurality of miRNAs that regulate the proliferation of cardiomyocytes, and have completed the present invention.

As a first aspect of the present invention, the present inventors provide miRNA having a cardiomyocyte proliferation promoting action. More specifically, the miRNA of the present invention includes the following embodiments:
(1-1) miRNA having a cardiomyocyte proliferation promoting action.
(1-2) The miRNA according to (1-1), wherein the miRNA is a substance selected from the group consisting of a mature miRNA, a precursor of the miRNA, a mutant thereof, and a similar substance.
(1-3) The miRNA according to (1-1) or (1-2) above, wherein the miRNA is a substance comprising a seed sequence consisting of SEQ ID NO: 1 or SEQ ID NO: 2.
(1-4) Substances containing a seed sequence consisting of SEQ ID NO: 1 are miR-148a (SEQ ID NO: 3), miR-148b (SEQ ID NO: 5), miR-152 (SEQ ID NO: 7 ) And their precursors, and their variants and similar substances, the miRNA according to (1-3) above.
(1-5) A substance comprising a seed sequence consisting of SEQ ID NO: 2 is a substance selected from the group consisting of miR-373 (SEQ ID NO: 9) and precursors thereof, and variants and similar substances thereof The miRNA according to (1-3) above.

  As a second aspect of the present invention, the present inventors have (a) a pharmaceutical composition for treating heart disease using a nucleic acid defining miRNA having the above-mentioned cardiomyocyte proliferation promoting action, ( b) a method of treating heart disease, characterized by introducing a nucleic acid defining miRNA having a cardiomyocyte proliferation promoting action into a damaged part of an individual's heart to proliferate the cardiomyocyte in the part, (c ) Use of a nucleic acid defining miRNA having a cardiomyocyte growth promoting action for the manufacture of a medicament for the treatment of heart disease, or (d) Cardiomyocyte growth promoting action for use in the treatment of heart disease Provided is an invention about a medicinal use of miRNA having a cardiomyocyte proliferation-promoting action, which can be described as a nucleic acid that defines miRNA. In the present invention, when referring to “nucleic acid defining miRNA”, (i) mature miRNA and precursor of the miRNA, and variants and similar substances thereof, or (ii) mature miRNA and precursor of the miRNA, and An expression vector for expressing these mutants and similar substances in vivo or in cells.

Regarding the invention of the pharmaceutical composition of the above-mentioned embodiment (a), more specifically, such a pharmaceutical composition includes the following embodiments:
(2-a-1) A pharmaceutical composition for treating heart disease, comprising a nucleic acid that defines miRNA having a cardiomyocyte proliferation promoting action.
(2-a-2) The miRNA described in (2-a-1) above, wherein the miRNA is a substance selected from the group consisting of a mature miRNA and a precursor of the miRNA, and variants and similar substances thereof Pharmaceutical composition.
(2-a-3) The miRNA is a substance comprising a seed sequence consisting of SEQ ID NO: 1 or SEQ ID NO: 2, according to (2-a-1) or (2-a-2) above Pharmaceutical composition.
(2-a-4) Substances containing a seed sequence consisting of SEQ ID NO: 1 are miR-148a (SEQ ID NO: 3), miR-148b (SEQ ID NO: 5), miR-152 (SEQ ID NO : The pharmaceutical composition according to (2-a-3) above, which is a substance selected from the group consisting of 7) and their precursors, and mutants and similar substances thereof.
(2-a-5) A substance comprising a seed sequence consisting of SEQ ID NO: 2 is selected from the group consisting of miR-373 (SEQ ID NO: 9) and its precursor, and its variants and similar substances The pharmaceutical composition according to the above (2-a-3), which is a substance.
(2-a-6) The above (2-a-1) to (2-), comprising a nucleic acid defining miRNA having a cardiomyocyte proliferation promoting action as an expression vector or liposome for introduction and expression in cardiomyocytes The pharmaceutical composition according to any one of a-5).
(2-a-7) The pharmaceutical composition according to (2-a-6) above, wherein the vector is a viral vector or a non-viral expression vector.
(2-a-8) virus vectors are adenovirus vectors, adeno-associated virus (AAV) vectors, retrovirus vectors, Japanese hemagglutinating virus (HVJ; aka Sendai virus) vectors, lentivirus vectors, vaccinia virus vectors, The pharmaceutical composition according to (2-a-7) above, which is selected from chicken poxvirus vectors and papovavirus vectors.
(2-a-9) The above (2-a-1), wherein the miRNA is a miRNA derivative comprising at least one modified internucleoside linkage, at least one modified sugar moiety, at least one modified base, or a combination thereof. ) To (2-a-8).
(2-a-10) The above (2-a-1) to (2-a-10), wherein the heart disease is myocardial infarction, ischemic heart disease, congestive heart failure, hypertrophic cardiomyopathy, dilated cardiomyopathy, myocarditis or chronic heart failure The pharmaceutical composition according to any one of (2-a-9).

Moreover, regarding the invention of the treatment method of (b) among the above-mentioned aspects, more specifically, such a treatment method includes the following aspects:
(2-b-1) A cardiovascular disease characterized by proliferating cardiomyocytes at an affected site of an individual's heart by introducing a nucleic acid defining miRNA having a cardiomyocyte proliferation promoting action into the damaged site of the individual's heart Method of treatment.
(2-b-2) The miRNA according to (2-b-1) above, wherein the miRNA is a substance selected from the group consisting of a mature miRNA and a precursor of the miRNA, and variants and similar substances thereof Method of treatment.
(2-b-3) The miRNA according to (2-b-1) or (2-b-2) above, wherein the miRNA is a substance comprising a seed sequence consisting of SEQ ID NO: 1 or SEQ ID NO: 2. Method of treatment.
(2-b-4) Substances containing a seed sequence consisting of SEQ ID NO: 1 are miR-148a (SEQ ID NO: 3), miR-148b (SEQ ID NO: 5), miR-152 (SEQ ID NO 7) The therapeutic method according to the above (2-b-3), which is a substance selected from the group consisting of 7) and their precursors, and mutants and similar substances thereof.
(2-b-5) A substance comprising a seed sequence consisting of SEQ ID NO: 2 is selected from the group consisting of miR-373 (SEQ ID NO: 9) and its precursor, and its variants and similar substances The treatment method according to (2-b-3) above, which is a substance.
(2-b-6) The above-mentioned (2-b-1) to (2), wherein a nucleic acid defining miRNA having a cardiomyocyte proliferation promoting action is introduced into the damaged part of the heart of an individual by an expression vector or liposome. The treatment method according to any one of -b-5).
(2-b-7) The method according to (2-b-6) above, wherein the vector is a viral vector or a non-viral expression vector.
(2-b-8) virus vectors include adenovirus vectors, adeno-associated virus (AAV) vectors, retrovirus vectors, Japanese hemagglutinating virus (HVJ; aka Sendai virus) vectors, lentivirus vectors, vaccinia virus vectors, The treatment method according to (2-b-7) above, which is selected from a chicken poxvirus vector and a papovavirus vector.
(2-b-9) The above (2-b-1), wherein the miRNA is a miRNA derivative comprising at least one modified internucleoside linkage, at least one modified sugar moiety, at least one modified base, or a combination thereof. ) To (2-b-8).
(2-b-10) (2-b-1) to (2-b-10) above, wherein the heart disease is myocardial infarction, ischemic heart disease, congestive heart failure, hypertrophic cardiomyopathy, dilated cardiomyopathy, myocarditis or chronic heart failure The treatment method according to any one of (2-b-9).

In addition, regarding the invention of use of (c) among the above-described embodiments, more specifically, such use includes the following embodiments:
(2-c-1) Use of a nucleic acid defining miRNA having a cardiomyocyte proliferation promoting action for the manufacture of a pharmaceutical composition for treating heart disease.
(2-c-2) The miRNA according to (2-c-1) above, wherein the miRNA is a substance selected from the group consisting of a mature miRNA and a precursor of the miRNA, and variants and similar substances thereof use.
(2-c-3) The miRNA is a substance comprising a seed sequence consisting of SEQ ID NO: 1 or SEQ ID NO: 2, according to (2-c-1) or (2-c-2) above use.
(2-c-4) Substances containing a seed sequence consisting of SEQ ID NO: 1 are miR-148a (SEQ ID NO: 3), miR-148b (SEQ ID NO: 5), miR-152 (SEQ ID NO : Use according to (2-c-3) above, which is a substance selected from the group consisting of 7) and their precursors, and mutants and similar substances thereof.
(2-c-5) The substance containing the seed sequence consisting of SEQ ID NO: 2 is selected from the group consisting of miR-373 (SEQ ID NO: 9) and its precursor, and its variants and similar substances The use according to (2-c-3) above, which is a substance.
(2-c-6) The above (2-c-1) to (2-), comprising a nucleic acid defining miRNA having a cardiomyocyte proliferation promoting action as an expression vector or liposome for introduction and expression in cardiomyocytes Use of any one of c-5).
(2-c-7) The use according to (2-c-6) above, wherein the vector is a viral vector or a non-viral expression vector.
(2-c-8) virus vectors include adenovirus vectors, adeno-associated virus (AAV) vectors, retrovirus vectors, Japanese hemagglutinating virus (HVJ; aka Sendai virus) vectors, lentivirus vectors, vaccinia virus vectors, The use according to (2-c-7) above, which is selected from a chicken poxvirus vector and a papovavirus vector.
(2-c-9) The above (2-c-1), wherein the miRNA is a miRNA derivative comprising at least one modified internucleoside linkage, at least one modified sugar moiety, at least one modified base, or a combination thereof. ) To (2-c-8).
(2-c-10) The above (2-c-1) to (2-c-10), wherein the heart disease is myocardial infarction, ischemic heart disease, congestive heart failure, hypertrophic cardiomyopathy, dilated cardiomyopathy, myocarditis or chronic heart failure Use of any one of (2-c-9).

Furthermore, regarding the nucleic acid invention of (d) among the above-described embodiments, more specifically, such nucleic acids include the following embodiments:
(2-d-1) A nucleic acid that defines miRNA having a cardiomyocyte proliferation promoting action for use in the treatment of heart disease.
(2-d-2) The miRNA according to (2-d-1) above, wherein the miRNA is a substance selected from the group consisting of a mature miRNA and a precursor of the miRNA, and variants and similar substances thereof Nucleic acid.
(2-d-3) The miRNA is a substance comprising a seed sequence consisting of SEQ ID NO: 1 or SEQ ID NO: 2, according to (2-d-1) or (2-d-2) above Nucleic acid.
(2-d-4) Substances containing a seed sequence consisting of SEQ ID NO: 1 are miR-148a (SEQ ID NO: 3), miR-148b (SEQ ID NO: 5), miR-152 (SEQ ID NO : The nucleic acid according to (2-d-3) above, which is a substance selected from the group consisting of 7) and their precursors, and mutants and similar substances thereof.
(2-d-5) A substance comprising a seed sequence consisting of SEQ ID NO: 2 is selected from the group consisting of miR-373 (SEQ ID NO: 9) and its precursor, and its variants and similar substances The nucleic acid according to (2-d-3), which is a substance.
(2-d-6) The above (2-d-1) to (2-), comprising a nucleic acid defining miRNA having a cardiomyocyte proliferation promoting action as an expression vector or liposome for introduction and expression in cardiomyocytes The nucleic acid according to any one of d-5).
(2-d-7) The nucleic acid according to (2-d-6) above, wherein the vector is a viral vector or a non-viral expression vector.
(2-d-8) virus vectors are adenovirus vectors, adeno-associated virus (AAV) vectors, retrovirus vectors, Japanese hemagglutinating virus (HVJ; aka Sendai virus) vectors, lentivirus vectors, vaccinia virus vectors, The nucleic acid according to (2-d-7) above, which is selected from a chicken poxvirus vector and a papovavirus vector.
(2-d-9) The above (2-d-1), wherein the miRNA is a miRNA derivative comprising at least one modified internucleoside linkage, at least one modified sugar moiety, at least one modified base, or a combination thereof. The nucleic acid according to any one of (2-d-8).
(2-d-10) The above (2-d-1) to (2-d-10), wherein the heart disease is myocardial infarction, ischemic heart disease, congestive heart failure, hypertrophic cardiomyopathy, dilated cardiomyopathy, myocarditis or chronic heart failure The nucleic acid according to any one of (2-d-9).

As a third aspect of the present invention, the present inventors provide a method for proliferating cardiomyocytes using the above-described nucleic acid that defines miRNA having a cardiomyocyte proliferation promoting action. More specifically, such cardiomyocyte proliferation methods include the following aspects:
(3-1) A method for proliferating cardiomyocytes, characterized by introducing a nucleic acid defining miRNA having a cardiomyocyte proliferation-promoting action into cardiomyocytes for expression.
(3-2) The method according to (3-1) above, wherein the miRNA is a substance selected from the group consisting of a mature miRNA, a precursor of the miRNA, a mutant thereof, and a similar substance.
(3-3) The method according to (3-1) or (3-2) above, wherein the miRNA is a substance comprising a seed sequence consisting of SEQ ID NO: 1 or SEQ ID NO: 2.
(3-4) Substances containing a seed sequence consisting of SEQ ID NO: 1 are miR-148a (SEQ ID NO: 3), miR-148b (SEQ ID NO: 5), miR-152 (SEQ ID NO: 7 ) And their precursors, and their variants and similar substances, the method according to (3-3) above.
(3-5) A substance comprising a seed sequence consisting of SEQ ID NO: 2 is a substance selected from the group consisting of miR-373 (SEQ ID NO: 9) and its precursors, and variants and similar substances thereof The method according to (3-3) above.
(3-6) Any of the above (3-1) to (3-5), comprising introducing a nucleic acid defining miRNA having a cardiomyocyte proliferation promoting action into cardiomyocytes using an expression vector or a liposome Or the method according to claim 1.
(3-7) The method according to (3-6) above, wherein the vector is a viral vector or a non-viral expression vector.
(3-8) Adenovirus vector, adeno-associated virus (AAV) vector, retrovirus vector, Japanese hemagglutinating virus (HVJ; aka Sendai virus) vector, lentivirus vector, vaccinia virus vector, chicken pox The method according to (3-7) above, which is selected from a viral vector and a papovavirus vector.
(3-9) The above (3-1) to (3), wherein the miRNA is a miRNA derivative comprising at least one modified internucleoside bond, at least one modified sugar moiety, at least one modified base, or a combination thereof. -8) The method according to any one of the above.

  By using the miRNA according to the present invention, a method for proliferating cardiomyocytes, a vector for treating heart disease, and a pharmaceutical composition for treating heart disease can be provided. The cardiomyocyte proliferation method according to the present invention can induce cardiomyocyte division and proliferate cells more efficiently than conventional methods. Cardiomyocytes prepared by the method can be used for various drugs. It can be used as a cell for screening and transplantation therapy. Moreover, by applying this method to gene therapy, the possibility of regenerative medical treatment of heart disease caused by necrosis or deletion of cardiomyocytes was obtained. It has been revealed that the miRNA according to the present invention exhibits proliferative activity remarkably against cardiomyocytes.

FIG. 1 shows in primary cultured cardiomyocytes transfected with miR-148a (SEQ ID NO: 3), miR-152 (SEQ ID NO: 7), miR-373 (SEQ ID NO: 9) in Example 2. It is a figure which shows a Ki67 positive cardiomyocyte rate. Ki67 is a cell nuclear protein and is a protein used as a marker for detecting proliferating cells. FIG. 2 shows infection of recombinant adenovirus expressing miR-148a (SEQ ID NO: 3), miR-152 (SEQ ID NO: 7), miR-373 (SEQ ID NO: 9) in Example 3. It is a figure which shows the Ki67 positive cardiomyocyte rate in the made primary cultured cardiomyocytes. None indicates unprocessed. FIG. 3-1 shows primary cultured myocardium transfected with miR-148a (SEQ ID NO: 3), miR-152 (SEQ ID NO: 7), and miR-373 (SEQ ID NO: 9) in Example 4. It is a figure which shows the phosphorylated histone H3 (H3P) positive cardiomyocyte rate in a cell. FIG. 3-2 shows a recombinant adenovirus expressing miR-148a (SEQ ID NO: 3), miR-152 (SEQ ID NO: 7), miR-373 (SEQ ID NO: 9) in Example 4. It is a figure which shows the H3P positive cardiomyocyte rate in the primary culture cardiomyocytes which infected A. None indicates unprocessed. FIG. 3-3 is a recombinant adenovirus expressing miR-148a (SEQ ID NO: 3), miR-152 (SEQ ID NO: 7), miR-373 (SEQ ID NO: 9) in Example 4. Is a photomicrograph of cells immunostained with anti-H3P antibody, anti-TroponinT antibody, and DAPI on primary cultured cardiomyocytes infected with. H3P: Green, TroponinT: Red, DAPI: Blue FIG. 4 shows the production of recombinant adenovirus expressing miR-148a (SEQ ID NO: 3), miR-152 (SEQ ID NO: 7), miR-373 (SEQ ID NO: 9) in Example 5. It is a figure which shows the Ki67 positive cardiomyocyte rate in the heart tissue section which infected the rat heart in the body.

  In practicing the present invention, for general methods such as molecular biology, microbiology, cell biology, and recombinant DNA technology and conventional techniques, those skilled in the art will use standard reference books in the field unless otherwise indicated. You can refer to it. These include, for example, Molecular Cloning: A Laboratory Manual 3rd edition (Sambrook & Russell, Cold Spring Harbor Laboratory Press, 2001); Current Protocols in Molecular biology (Ausubel et al., John Wiley & Sons, 1987); Methods in Enzymology Series (Academic Press); PCR Protocols: Methods in Molecular Biology (Bartlett & Striling, Humana Press, 2003); Animal Cell Culture: A Practical Approach 3rd Edition (Masters, Oxford University Press, 2000); Antiboides: A Laboratory See Manual (Harlow et al & Lane, Cold Spring Harbor Laboratory Press, 1987). In addition, the reagents and kits for cell culture and cell biology experiments referred to in this specification are commercially available (from Sigma, Aldrich, Invitrogen / GIBCO, Clontech, Stratagene, etc.). It can be obtained.

  As a first aspect, the present invention provides miRNA having a cardiomyocyte proliferation promoting action.

  The present invention provides, as a second aspect, a pharmaceutical composition for treating a heart disease using the above-described nucleic acid that defines miRNA having a cardiomyocyte proliferation promoting action; miRNA having a cardiomyocyte proliferation promoting action A method for treating a heart disease, comprising introducing a nucleic acid that defines the above into a damaged site of a heart of an individual to proliferate cardiomyocytes at the site; myocardium for producing a pharmaceutical composition for treating a heart disease Also provided is the use of a nucleic acid defining a miRNA having a cell growth promoting effect; and a nucleic acid defining a miRNA having a cardiomyocyte growth promoting effect for use in the treatment of heart disease.

  As a third aspect, the present invention provides a method for proliferating cardiomyocytes using the above-described nucleic acid that defines miRNA having a cardiomyocyte proliferation promoting action.

Cardiomyocytes Cardiomyocytes to be propagated using the method according to the present invention may generally include adult cardiomyocytes, juvenile cardiomyocytes, and cardiomyocytes at all stages of development of fetal cardiomyocytes. And in the present invention, cardiomyocytes present in the heart tissue of mammals can be directly targeted, but in the first and third aspects of the present invention, cultured cardiomyocytes in vitro Etc. can also be used.

  In the present invention, any cardiomyocytes derived from any source can be used as cultured cardiomyocytes in vitro. For example, cardiomyocytes isolated from living heart tissue by various methods such as enzyme treatment Alternatively, primary cultured cells obtained by culturing them under appropriate culture conditions for about 1 to 5 days can also be used. Specific methods for culturing cardiomyocytes are described in many references, but representative methods include Chien's method and its modified methods (Chien et al., The Journal of Clinical Investigation, 75: 1770- 1780, (1985); Meidell et al., American Journal of Physiology, 251: H1076-H1084, (1986); Tamamori et al., American Journal of Physiology, 275: H2036-H2040, (1998)) be able to.

  Further, the cultured cardiomyocytes are not limited to the above examples, and also include cardiomyocytes obtained by inducing differentiation from various stem cells. The stem cell used in the practice of the present invention means a cell having a property capable of differentiating into a cell having a cardiomyocyte-like character under in vitro culture. Specifically, it is already widely used as a cultured cell. Examples include embryonic stem cells (ES cells) derived from mammals such as mice, monkeys and humans, pluripotent stem cells such as artificial pluripotent stem cells (iPS cells) and embryonic germ cells (EG cells). A standard protocol has already been established for the production, passage, and storage of these cells, and those skilled in the art will recognize several reference books such as Manipulating the Mouse Embryo: A laboratory manual (Hogan et al., Cold Spring Harbor Laborayory Press, 1994), Embryonic Stem Cells (Turksen, Humana Press, 2002), and several references (Matsui et al., Cell, 70: 841-847, (1992); Shamblott et al., Proceedings of the National Academy of Sciences of the United States of America, 95: 13726-13731, (1998); Okita et al., Nature, 448: 313-317, (2007); Takahashi et al., Cell, 131: 861-872, (2007)), these pluripotent stem cells can be easily used. The stem cells that can be used in the present invention are not limited to the above-described stem cells, and any stem cells having a trait similar to ES cells can be used. In this case, ES cell-like traits include the presence of ES cell-specific surface (antigen) markers, the expression of ES cell-specific genes, and the ability to form teratoma and chimeric mice. It can be defined with cell biology specific to ES cells.

  In addition, even cells that do not have traits similar to ES cells or cells that are not pluripotent stem cells should be capable of differentiating into cells having cardiomyocyte-like traits at least in vitro culture. For example, the method described in the present invention can be used. Examples of such cells include bone marrow mesenchymal stem cells derived from bone marrow cells (Bruder et al., USP 5,736,396; Pittenger et al., Science, 284: 143-147, (1999)) and CMG cells (Makino et al. al., The Journal of Clinical Investigation, 103: 697-705, (1999); WO01 / 048151), Spoc cells derived from muscle tissue (WO03 / 035382), and the like.

  In the present invention, any culture method can be used as a culture method for producing cardiomyocytes from stem cells as long as the method is suitable for inducing differentiation of cardiomyocytes. Regarding specific differentiation induction methods, a plurality of methods have already been established. For those skilled in the art, for example, a reference book such as Embryonic Stem Cells (Turksen, Humana Press, 2002) and a plurality of references (Klug et al., The Journal of Clinical Investigation, 98: 216-224, (1996); Wobus et al., Journal of Molecular and Cellular Cardiology, 29: 1525-1539, (1997); Kehat et al., The Journal of Clinical Investigation, 108: 407-414, (2001); Xu et al., Circulation Research, 91: 501-508, (2002); Takahashi et al., Circulation, 107: 1912-1916, (2003); Cardiomyocytes can be induced to differentiate from stem cells by referring to Field et al., USP 6,015,671; Xu et al., WO03 / 06950).

MicroRNA (miRNA)
The present invention provides a microRNA (miRNA) having an effect of promoting proliferation of cardiomyocytes. The miRNA according to the present invention may be any miRNA that promotes the proliferation of cardiomyocytes, and can be used for growing the cardiomyocytes.

  In the present invention, miRNA is a small RNA that does not encode a protein that is highly expressed in eukaryotes including mammals, and is considered to be involved in various life phenomena such as development, differentiation, and proliferation. ing. The miRNA that can be used in the present invention includes mature miRNA and precursors of the miRNA, and mutants and similar substances thereof. The miRNA according to the present invention may be single-stranded or double-stranded.

  In the present invention, a mature miRNA is a fully processed miRNA and is 15 to 30 nucleotides long (mostly 21 to 25 nucleotides long). Mature miRNAs share at least 6 consecutive nucleotides from 1 to 12 nucleotides 5 ′ of the mature miRNA molecule (the nucleotide portion is also referred to as the “seed region” (Brennecke et al., PLoS Biology, 3: e85, (2005)), which can constitute a miRNA family consisting of a group of miRNAs, so the 5 'region of mature miRNA is identical to the miRNA family, but the 3' Different mature miRNAs are included, and if they belong to the same miRNA family, they may have the same function even if the nucleic acid sequence of the 3 'region is different. The 8-mer seed region sequence (seed sequence) has been reported (Brennecke et al., PLoS Biology, 3: e85, (2005)), and in this case, A sequence complementary to the binding sequence of the mer seed region is shared with the 5 'region. It has been reported that two different miRNAs in the miRNA constituting the miRNA family possessed can suppress the expression of a reporter gene containing an 8-mer target, although the 3 'side is different.

  The precursor of miRNA is a precursor RNA of mature miRNA. In the process of producing mature miRNA, miRNA is first transcribed into miRNA primary transcript (pri-miRNA) of several hundred to several thousand bases from miRNA gene present on genomic DNA, It is processed in the nucleus by an RNase III enzyme called Drosha, and becomes a precursor miRNA (pre-miRNA) having a hairpin structure of about 70 bases. Therefore, in the present invention, miRNA precursor includes miRNA primary transcript (pri-miRNA) and precursor miRNA (pre-miRNA).

  A miRNA variant is a miRNA in which one or several nucleotides are deleted, substituted, inserted or added as compared with a mature miRNA or a precursor of a mature miRNA originally possessed by a living body. Here, the term “several” means, for example, 7 to 6, preferably 5 to 4, and more preferably 3 to 1. As described above, there is a “seed region” on the 5 ′ side of the mature miRNA molecule that constitutes the miRNA family, and if the nucleic acid sequence of the 3 ′ side region is different as long as it belongs to the same miRNA family, it is the same It has the function of. Therefore, the miRNA mutant is preferably an miRNA in which nucleotides are deleted, substituted, inserted or added in a portion other than the seed region of the mature miRNA.

  An miRNA analog is an miRNA synthesized to mimic an endogenous mature miRNA or a precursor of mature miRNA. Such materials are commercially available (eg, from Ambion) and are readily available.

  In order to enhance the stability of the miRNA of the present invention, for example, a nucleic acid derivative and / or a nucleic acid having a modified internucleoside bond is used. These nucleic acid derivatives can be appropriately selected from known ones. Specifically, for example, methylphosphonate, phosphorothioate, phosphorodithioate, phosphoramidate, phosphotriester, sulfone, siloxane, Carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridge Bridged phosphorothioate, dimethylene-sulfide, dimethylene-sulfoxide, dimethylene-sulfone, 2'-O-alkyl, and 2'-deoxy-2'-fluor Examples include nucleic acids containing internucleoside linkages of 2'-deoxy-2'-fluoro phosphorothioate (Uhlmann & Peyman, Chemical Reviews, 90: 543-584, (1990), Schneider et al., Tetrahedron Letters, 31: 335-338, (1990)). Furthermore, derivatives such as Locked Nucleic Acid (Koshkin et al., Journal of the American Chemical Society, 120: 13252-13253 (1998)) and ENA (US7335765) are also included. Such a nucleic acid derivative and / or miRNA having a modified internucleoside bond is referred to as a miRNA derivative in the present invention. The miRNA derivatives include mature miRNA derivatives, miRNA precursor derivatives, miRNA variant derivatives, and miRNA analog derivatives.

  The miRNA that can be used in the present invention is obtained, for example, by isolating it from a natural product, chemically synthesizing it, or by producing it by a gene recombination technique using a known technique. It can also be obtained as a commercial product. For example, mature miRNA is registered in Sanger miRbase, and all the registered miRNAs can be obtained from Ambion, Qiagen, Sigma, Thermo Scientific, and the like.

  As a means for delivering miRNA into cells in a living body or cells in vitro, any of gene delivery means commonly used in the art, such as expression vectors or liposomes, should be used. Can do. The above mature miRNA or miRNA precursor can be used in the form of oligonucleic acid or derivative, but from the viewpoint of its expression efficiency and duration of effect, it should be used in the form incorporated into an expression vector. Is preferred.

  When an expression vector is used as a gene delivery means, any expression vector may be used so long as it has a promoter capable of expressing miRNA, and a viral expression vector or a non-viral expression vector may be used. it can.

  Viral expression vectors include adenovirus, adeno-associated virus (AAV), retrovirus, Japanese hemagglutinating virus (HVJ; also known as Sendai virus), lentivirus, vaccinia virus, chicken poxvirus, and papovaviruses such as SV40 Vectors derived from the above are exemplified, but preferably, by using an adenovirus vector, AAV vector, HVJ vector, or lentivirus vector, efficient gene transfer and high-level expression of the introduced gene are performed. Can do.

  Gene transfer with these viral vectors is one of the most powerful methods of introducing genes into mammalian cells and is used to introduce into virtually all types of human cells and many non-human cells it can.

  Moreover, since infection with these viruses does not depend on the cell cycle, genes can be expressed in various primary culture cell lines and transformed cell lines. In particular, genes can be efficiently introduced into cells that do not undergo DNA synthesis or cell division, such as cardiomyocytes. Since many cells receive multiple copies of recombinant DNA (or RNA) after infection, the introduced gene is temporarily expressed at a high level.

  In the case of adenovirus vectors, HVJ vectors, etc., DNA / RNA usually stays in the cytoplasm and is not taken up by the nucleus. Therefore, when these viral vectors are used, there is also an advantage that almost no mutagenic error that randomly occurs when a foreign gene is integrated into the host cell genome.

  The adenovirus vector used as one of the preferred forms of the present invention is a method using homologous recombination using human fetal kidney 293 cells or E. coli as a host (Miyake et al., Proceedings of the National Academy of Sciences of the United States). States of America, 93: 1320-1324, (1996)) or a simple in vitro ligation method (Mizuguchi & Kay, Human Gene Therapy, 9: 2577-2583, (1998)).

  Adenovirus is one of DNA viruses having double-stranded DNA genome, and human adenovirus type 5 and type 2 are the best studied. By deleting most of the E1 and E3 genes of these wild-type adenoviruses, it is possible to create viral vectors that are not capable of replication and have several kb of foreign sites without adversely affecting virus particle formation. It becomes possible to insert DNA. The recombinant adenovirus lacks the E1 gene, which is a transcriptional regulator, but is inserted by the inserted gene-specific transcription unit without depending on the growth of target cells or the presence or absence of other viral genes. Only the target gene can be expressed.

  Adeno-associated virus (AAV) is a single-stranded DNA virus of about 4.7 kb belonging to the Parvoviridae family, and serotypes AAV of type 1 to 12 are known. The AAV vector is made by replacing Rep (involved in replication and integration) and Cap (capsid) between the ITR (Inverted terminal repeat) at both ends with the target gene. This vector is introduced into 293 cells together with plasmids expressing Rep and Cap, and further adenovirus is infected as a helper virus to prepare a recombinant adeno-associated virus vector accumulated in the nucleus. Recently, a method has been developed that does not require a helper virus by introducing E2A, E4, and VA-expressing plasmids into 293 cells that express E1A and E1B.

  Sendai virus (SeV) is a rodent virus belonging to the genus Paramyxovirus, and is also referred to as a Japanese hemagglutinating virus (HVJ). The SeV genome consists of negative single-stranded RNA (complementary to mRNA), nucleoprotein (NP), nucleoprotein kinase (P), virus surface-lined protein (M), hemagglutinating protein (HN), membrane fusion It encodes 6 genes: protein (F) and RNA synthase (L). SeV binds to the sialic acid of the host cell by HN (hemaglutination-neuraminidase) protein and binds to it, and the genome is injected into the cytoplasm. The viral genome that has migrated into the cell transcribes and replicates the gene by the L protein that it encodes. Unlike normal viruses, this step is performed in the cytoplasm and the viral genome is not transferred into the nucleus.

  SeV vectors can be created as non-transmissible vectors by deleting one or more F, HN, and M genes other than the N, P, and L genes required for autonomous replication of the vector in the introduced cell. . F gene-deficient vectors include plasmids with recombinant vector cDNA inserted downstream of the T7 promoter recognized by bacteriophage T7-derived RNA polymerase, N, P, F, and L expression plasmids under T7 promoter control, and T7 RNA It is produced by introducing a recombinant vaccinia virus expressing polymerase into monkey kidney-derived LLC-MK2 cells. Recently, six plasmids (a plasmid with a recombinant vector cDNA inserted downstream of the T7 promoter and a CAG promoter) A simple production method by only transfection of N, P, L, F, and T7 RNA polymerase gene expression plasmids under control is also possible.

For a review of adenoviral vectors and other viral vectors, as well as methods of production and use, one skilled in the art can refer to multiple reference books. For example, Gene Therapy Protocols: Methods in Molecular Medicine (Robbins, Humana Press, 1997), Gene Transfer in Cardiovascular System: Experimental Approaches & Therapeutic Implications (March, Kluwer Academic Publishers, 1997), Adenoviral Vectors for Gene Therapy (Curiel & Douglas, Academic Press, 2002). Kits for producing adenovirus vectors are also commercially available (from Invitrogen's ViraPower ^™ adenovirus expression system (# K4930), Takara Bio's Adenovirus Expression Vector Kit (# 6170), etc.) and are readily available. Can be used to implement the present invention.

  Kits for preparing AAV vectors and SeV vectors are also commercially available (AAV Helper-Free system (Stratagene; # 240071), recombinant Sendai virus mini vector preparation kit (MBL; DV-0001), etc.) Are readily available and can be used to implement the present invention.

  Non-viral expression vectors include, for example, promoters derived from viruses such as SV (Simian virus) 40 virus promoter, cytomegalovirus (CMV) promoter, Rous sarcoma virus promoter, β-actin CAG promoter (Niwa et al.) Which is a hybrid promoter in which a CMV enhancer and a poly A addition signal sequence of a rabbit β-globin gene are incorporated into an avian β-actin promoter such as a promoter, EF (elongation factor) 1α promoter , Gene, 108: 193-199, (1991)), and vectors having U6 or H1 promoter which is a Pol.III promoter, but are not limited to these vectors. Expression vectors that can use these promoters are commercially available (from Ambion, Invitrogen, TAKARA BIO, etc.) and can be easily obtained.

  As a method for introducing the above gene or gene vector, all known methods can be used. For example, a transfection method using calcium phosphate or an electric pulse, a cell after encapsulating a target gene in a liposome or the like. And a method in which a gene of interest is incorporated into a virus vector such as a retrovirus or an adenovirus, and a cell is infected with the recombinant virus. In this case, the viral vector is a construct that can incorporate and express a target gene in a nucleic acid sequence in which the entire length or part of viral DNA or RNA is deleted or mutated.

  Specific examples of liposomes include lipid preparations containing N- [2,3- (dioleoyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA) and dioleoylphosphatidylethanolamine (DOPE). Can be used.

Acquisition of specific miRNAs The present inventors searched a library of mature miRNAs based on whether or not they exhibited a remarkable BrdU uptake ability when introduced into cardiomyocytes. As mature miRNAs having an action, miR-148a (SEQ ID NO: 3), miR-148b (SEQ ID NO: 5), miR-152 (SEQ ID NO: 7), and miR-373 (SEQ ID NO: 9) etc. were found and used for further experiments.

  Preferred miRNAs that can be used in the present invention also include precursors of these mature miRNAs. For example, as a precursor of mature miRNA, miRNA primary transcript (pri-miRNA) or precursor miRNA (pre-miRNA) of each miRNA can be mentioned as an example. Specific examples thereof include, for example, the following primary transcripts (pri-miRNAs) of genes encoding the specific mature miRNAs described above, and preferred miRNAs that can be used in the present invention. Mir-148a (precursor of miR-148a, SEQ ID NO: 4), mir-148b (precursor of miR-148b, SEQ ID NO: 6), mir-152 (precursor of miR-152, SEQ ID NO: 8), and mir-373 (precursor of miR-373, SEQ ID NO: 10).

Examples of miRNA that can be used in the present invention include mutants and similar substances of mature miRNA and precursor miRNA shown in Table 1 above, or precursors of miRNA other than the precursor of miRNA shown in Table 1 above. The body (eg, miRNA primary transcript (pri-miRNA) and the like) and variants and similar substances thereof are also included.

  For example, miR-148a (SEQ ID NO: 3), miR-148b (SEQ ID NO: 5) and miR-152 (SEQ ID NO: 7), which are mature miRNAs that can be used in the present invention, are respectively Has a common seed sequence, ucagugca (SEQ ID NO: 1), on the 5 ′ side of the miRNA and constitutes the miR-148 family. The sequences of miRNAs belonging to the miR-148 family are listed below. Underline the stored seed sequence.

miR-148a: ucagugca cuacagaacuuugu (SEQ ID NO: 3)
miR-148b: ucagugca ucacagaacuuugu (SEQ ID NO: 5)
miR-152: ucagugca ugacagaacuugg (SEQ ID NO: 7).

  Therefore, as one of the miRNAs of the present invention, a mature miRNA containing a seed sequence consisting of ucagugca (SEQ ID NO: 1) at the 5 ′ end or a precursor of the miRNA, or a variant or a similar substance thereof is included. . These miRNAs include miR-148a (SEQ ID NO: 3), miR-148b (SEQ ID NO: 5), miR-152 (SEQ ID NO: 7) and precursors of the miRNA, and variants thereof And miRNA selected from the group consisting of similar substances. For example, precursors of mature miRNAs miR-148a (SEQ ID NO: 3), miR-148b (SEQ ID NO: 5), and miR-152 (SEQ ID NO: 7) include pri of each miRNA. -miRNA, pre-miRNA and the like. Specific examples thereof include precursors represented by the following SEQ ID NOs: 4, 6 and 8, respectively.

Human mir-148a: gaggcaaagu ucugagacac uccgacucug aguaugauag aagucagugc acuacagaac uuugucuc (SEQ ID NO: 4)
Human mir-148b: caagcacgau uagcauuuga ggugaaguuc uguuauacac ucaggcugug gcucucugaa agucagugca ucacagaacu uugucucgaa agcuuucua (SEQ ID NO: 6)
Human mir-152: uguccccccc ggcccagguu cugugauaca cuccgacucg ggcucuggag cagucagugc augacagaac uugggcccgg aaggacc (SEQ ID NO: 8).

  Another mature miRNA that can be used in the present invention, miR-373, has a common seed sequence, gaagugcu (SEQ ID NO: 2), on the 5 ′ side of the miRNA, and miR-373 Make up a family. The sequence of miRNA belonging to the miR-373 family is shown. The seed array is underlined.

miR-373: gaagugcu ucgauuuuggggugu (SEQ ID NO: 9).

  Accordingly, as one of the miRNAs of the present invention, a mature miRNA containing a seed sequence consisting of gaagugcu (SEQ ID NO: 2) at the 5 ′ end, a precursor of the miRNA, or a mutant or similar substance thereof is included. . These miRNAs include miR-373 (SEQ ID NO: 9) and precursors of the miRNA, as well as miRNAs selected from the group consisting of variants and analogs thereof. For example, examples of the precursor of miR-373 (SEQ ID NO: 9), which is a mature miRNA, include pri-miRNA and pre-miRNA of the miRNA. Specific examples thereof include a precursor represented by the following SEQ ID NO: 10.

  Human miR-373: gggauacuca aaaugggggc gcuuuccuuu uugucuguac ugggaagugc uucgauuuug ggguguccc (SEQ ID NO: 10).

It was clarified that the miRNA according to the present invention obtained in this way has a proliferative activity remarkably against cardiomyocytes.
The miRNA according to the present invention includes MRC-5 (human fetal lung-derived) cells, WI-38 (human fetal lung-derived) cells, MC3T3-E1 (mouse calvarial-derived) cells and the like, which are normal cell lines. Did not show significant growth-promoting activity.

Application as a gene therapy method As another aspect of the present invention, an expression vector such as a viral vector or a non-viral vector, which is used in the practice of the present invention, includes a nucleic acid that defines miRNA having a cardiomyocyte proliferation promoting action. There is provided a pharmaceutical composition for gene therapy, comprising a vector (hereinafter simply referred to as “gene expression vector”), preferably a viral vector, more preferably an adenoviral vector or HVJ vector, AAV vector, lentiviral vector and the like. The nucleic acid that defines the miRNA contained in the gene expression vector may be any nucleic acid that defines the miRNA described in the section “Acquisition of specific miRNA” described above. Since this pharmaceutical composition for gene therapy contains a nucleic acid that defines miRNA having a cardiomyocyte proliferation promoting action, it can be used as a myocardial regenerative drug or a cardiac disease therapeutic drug by the action of miRNA. The heart disease to be treated is not particularly limited as long as it is accompanied by weakness, cessation or death of cardiomyocytes. Specific examples include myocardial infarction, ischemic heart disease, congestive heart failure, hypertrophy Cardiomyopathy, dilated cardiomyopathy, myocarditis, chronic heart failure and the like.

  The dosage form of the pharmaceutical composition is not particularly limited, and it can be formulated by a conventional method. For example, it may be in the form of an injectable preparation containing the gene expression vector of the present invention in a pharmaceutically acceptable drug carrier or diluent such as sterile water or buffered saline. The drug carrier can also include other suitable stabilizers (eg, nuclease inhibitors, etc.), chelating agents (eg, EDTA, etc.), and / or other auxiliaries. The pharmaceutical composition containing the above components can be sterilized by a method such as filtration as necessary and filled in a container such as a sterile ampoule.

The dosage of the pharmaceutical composition of the present invention should be appropriately increased or decreased depending on conditions such as the patient's age, sex, body weight, symptoms, and administration route. It can be set as appropriate. Generally, the amount of active ingredient DNA per adult is in the range of about 1.0 μg / kg to 1.0 g / kg, preferably in the range of about 10 μg / kg to 100 mg / kg. When a viral vector such as an adenoviral vector is used, the final titer of the virus is preferably 10 ⁷ to 10 ¹³ pfu / mL, more preferably 10 ⁹ to 10 ¹² pfu / mL.

  The pharmaceutical composition of the present invention may be supplied as a complex with a liposome, particularly when the gene expression vector is based on a non-viral vector. With such a configuration, there is a possibility that high transfection efficiency can be realized particularly in cardiomyocytes. Specific examples of liposomes include many new ones including N- [2,3- (dioleoyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA) and dioleoylphosphatidylethanolamine (DOPE). Lipid formulations have been developed and tested for transfection using various cell lines (Banerjee, Journal of Biomaterials Applications, 16: 3-21, (2001); Maurer et al., Expert Opinion on Biological Therapy, 1: 923-947, (2001)). Also, a method using a fusion-forming virus liposome having a fusion-forming envelope derived from HVJ, the so-called HVJ-liposome method (Yonemitsu et al., International Journal of Oncology, 12: 1277-1285, (1998); Kaneda et al ., Molecular Medicine Today, 5: 298-303, (1999)) is also effective. Liposomes for the introduction of these nucleic acids are commercially available. Especially for miRNA introduction, Lipofectamine RNAi MAX (Invitrogen), Oligofectamine (Invitrogen), siLentFect (BIO-RAD), HiPerFect (Qiagen) can be used.

  The above gene expression vector or a pharmaceutical composition containing the gene expression vector may be introduced into the entire heart of a heart disease patient, but it is preferable to introduce the gene expression vector in a limited area. In the present invention, the site of injury refers to a site where a cardiomyocyte in an individual (human or non-human animal: the same applies hereinafter) is weakened, ceases to function or has died, and its nearby region, or is weakened in the individual, progression of functional death or death. Means the expected site. In this case, as a method of introducing a gene expression vector or a pharmaceutical composition containing the same into an injured site, it is possible to continuously transport the preparation to the injured site using an osmotic pump, an osmotic tube, etc. Examples include a method of injecting directly into the heart using a syringe after opening the chest, a method of injecting transvascularly (indirectly) using a catheter under fluoroscopy, and the like.

  The transvascular method is preferable because gene transfer can be confined to the heart. In this case, the gene expression vector or a pharmaceutical composition containing the gene can be injected into the blood vessel and transported to the cardiomyocytes via the bloodstream. It can also be injected directly into the myocardium and contacted with cardiomyocytes. Surgical techniques using such catheters are known in the art, and as reference books, for example, Gene Transfer in Cardiovascular System: Experimental Approaches & Therapeutic Implications (March, Kluwer Academic Publishers, 1997), Examples include books such as Vascular Surgery 5th edition (Rutherford, WB Saunders, 2000), Textbook of Interventional Cardiology 4th edition (Topol, WB Saunders, 2002). In addition, catheters used for carrying out the above method are commercially available (from Boston Scientific, Edwards Lifesciences Corporation, etc.) and can be easily obtained.

Utilization of cardiomyocytes proliferated by the method of the present invention Cardiomyocytes proliferated by the method of the present invention can be efficiently converted to highly purified cardiomyocytes by using cell recovery, separation and purification methods by known methods. And can be obtained in large amounts. The cardiomyocytes thus obtained are hereinafter referred to as cardiomyocytes prepared according to the present invention.

  Any known cell separation and purification method can be used for the purification of cardiomyocytes. Specific examples thereof include antigen-antibody reactions such as flow cytometers, magnetic beads, and panning methods. An equivalent method and a cell fractionation method by density gradient centrifugation using a carrier such as sucrose or percoll can be exemplified.

  In addition, as another cardiomyocyte sorting method, a modification such as artificially imparting drug resistance or ectopic protein expression ability to the gene of stem cells such as animal cells or ES cells to be a cardiomyocyte source in advance, A method of selectively collecting cardiomyocytes based on these indices is mentioned. For example, Field and co-workers have introduced a gene cassette capable of expressing a neomycin (G418) resistance gene under the control of an α-type myosin heavy chain promoter into mouse ES cells so that the ES cells become cardiomyocytes. A system that can survive in a medium supplemented with G418 only when it has differentiated and expressed the α-type myosin heavy chain gene, and the cells selected as G418-resistant cells by this method have a probability of 99% or higher. (USP 6,015,671; Klug et al., The Journal of Clinical Investigation, 98: 216-224, (1996)).

  As another aspect of the present invention, the cardiomyocytes prepared according to the present invention are useful for pharmacological evaluation and activity evaluation of various physiologically active substances (for example, drugs) and novel gene products whose functions are unknown. For example, it can be used for screening for substances and drugs related to cardiomyocyte function regulation, or substances and drugs that are toxic or toxic to cardiomyocytes. In a further aspect, an evaluation kit comprising cardiomyocytes prepared according to the present invention is useful for the screening.

  The test substance to be used for screening may be any kind as long as it can be added to the culture system, for example, low molecular weight compounds, high molecular weight compounds, organic compounds, inorganic compounds, proteins, peptides, genes, viruses, cells, cell cultures. Liquid, microbial culture solution, and the like. Examples of a method for efficiently introducing a gene into a culture system include a method for adding to a culture system using a viral vector such as a retrovirus or adenovirus, or a method for encapsulating in a liposome or the like and adding to a culture system. .

  The test substance can be evaluated by measuring qualitative or quantitative changes in cardiomyocyte function. Taking cardiomyocyte viability as an example, cardiomyocytes prepared according to the present invention are seeded on a culture plate to an appropriate cell density, and cultured in a serum-free medium to induce cell death (apoptosis). In this case, an appropriate amount of the test substance may be added to the medium, and the survival rate or mortality of the cardiomyocytes may be measured. As a method for measuring the survival rate or mortality of cardiomyocytes, it may be based on macroscopic observation using dye uptake such as trypan blue, or a method using dehydrogenase activity (reduction activity) as an index, Furthermore, a method using caspase activity specific to apoptotic cells or annexin V expression as an index may be used. Kits using this mechanism are commercially available (from Sigma, Clonetech, Promega, etc.) and can be easily obtained.

  Substances and drugs obtained by the above screening methods have cardiomyocyte differentiation-inducing action and function-regulating action. For example, myocardial infarction, ischemic heart disease, congestive heart failure, hypertrophic cardiomyopathy, dilated cardiomyopathy, cardiomyopathy It can be used as a preventive or therapeutic agent for heart diseases such as inflammation and chronic heart failure. These compounds may be novel compounds or known compounds.

  The cardiomyocytes prepared according to the present invention can be used as transplanted cells for myocardial regeneration or heart disease treatment. Examples of the heart disease include myocardial infarction, ischemic heart disease, congestive heart failure, hypertrophic cardiomyopathy, dilated cardiomyopathy, myocarditis, chronic heart failure and the like. As the cells for transplantation, if the cardiomyocytes prepared according to the present invention are contained in high purity, the cells are suspended in an aqueous carrier such as a medium, and the cells are encapsulated in a solid phase carrier such as a biodegradable substrate. Any shape can be used, such as a buried or processed cardiomyocyte sheet (Shimizu et al., Circulation Research, 90: e40-e48, (2002)).

  Methods for transplanting the above-mentioned cardiomyocytes for transplantation into the injured site include a method in which the chest is opened and injected directly into the heart using a syringe, a method in which a part of the heart is surgically incised, and a catheter. Transplantation by the transvascular method used (Murry et al., Cold Spring Harbor Symposia on Quantitative Biology, 67: 519-526, (2002); Menasche, The Annals of Thoracic Surgery, 75: S20-S28, ( 2003); Dowell et al., Cardiovascular Research, 58: 336-350, (2003)), but is not particularly limited. It has been reported that when cardiomyocytes recovered from fetal heart are transplanted into the heart of a heart-impaired animal by such a method, a very good therapeutic effect is shown (Menasche, The Annals of Thoracic Surgery, 75: S20-S28, (2003); Reffelmann et al., Heart Failure Reviews, 8: 201-211, (2003)). ES cell-derived cardiomyocytes exhibit very similar traits to those derived from fetal heart (Maltsev et al., Mechanisms of Development, 44: 41-50, (1993); Maltsev et al., Circulation Research, 75: 233-244, (1994)). In addition, it has also been confirmed that animal experiment examples in which ES cell-derived cardiomyocytes were actually transplanted into an adult heart showed extremely high engraftment, which is almost the same as in cases in which fetal myocardium was transplanted (Klug et al. , The Journal of Clinical Investigation, 98: 216-224, (1996)). Therefore, in the above heart diseases caused by exhaustion and loss of cardiomyocytes, it is possible to promote improvement of cardiac function by supplementarily transplanting the cardiomyocytes prepared by the method described in the present invention into the pathological heart tissue. I can expect.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated more concretely, a following example shows only the illustration of this invention, and does not limit the scope of the present invention at all.

Example 1: Screening of miRNA library using BrdU incorporation as an index using primary cultured cardiomyocytes 2 to 4 days after birth (Wistar strain, obtained from Shimizu Experimental Materials Co., Ltd.) hearts were extracted and treated with collagenase Percoll concentration gradient centrifugation from the obtained cell group (percol solution with a density of 1.082 g / mL in which cells were suspended was layered on a percoll solution with a density of 1.050 g / mL and 1.060 g / mL, and 4 ° C, 3000 rpm (2000 G) The cardiomyocyte fraction was collected by centrifugation for 25 minutes. The cardiomyocytes obtained in this way were suspended in Eagle's minimum medium (Nacalai Tesque) supplemented with 5% calf serum (FCS; SAFC Bioscience), and then seeded in a culture dish. In a carbon dioxide incubator, Cultured at 37 ° C. Pre-miR ^™ miRNA Precursor Library-HumanV2 (Ambion: miRBase Sequence Database Version 8.0) 328 human mature miRNAs were prepared from cardiomyocytes prepared in this way (15,000 cells / well per 96-well plate). Library designed to mimic) Transfect 2 pmol of each using Lipofectamine ^TM RNAiMAX (Invitrogen) and measure BrdU uptake 2 days later using cell proliferation ELISA and BrdU chemiluminescence kit (Roche) Compared with the amount of BrdU incorporation in the negative control group, the Pre-miR ^TM miRNA Precursor Molecule-Negative control # 1 (Ambion) addition group, Was selected as a candidate miRNA that significantly promotes. In this test, miR-148a, miR-148b, miR-152, and miR-373 could be selected as examples of the candidate miRNA.

Example 2: Analysis of cell cycle progression marker by forced miRNA expression in primary cultured cardiomyocytes For candidate cardiomyocytes (100,000 cells / well per 24-well plate), candidate miRNAs obtained in Example 1, miR-148a, miR Pre-miR ^TM miRNA Precursor Molecule designed to mimic human mature miRNAs -148b, miR-152 and miR-373 and negative control Negative control # 1 (all Ambion) 1 pmol Lipofectamine ^TM RNAiMAX (Invitrogen ) And cells were fixed with 4% paraformaldehyde 2-4 days after transfection.

Next, after anti-Ki67 antibody (ThermoSCIENTIFIC) (1: 200 dilution) and anti-TroponinT antibody (Thermo SCIENTIFIC) (1: 200 dilution) were reacted, Alexa Fluor ^TM labeled antibodies (Alexa-488 and Alexa-568; Molecular Probes ) (Both 1: 400 dilution). The cell nuclei were stained with a 4 ', 6-diamidine-2'-phenylindole dihydrochloride (DAPI) solution (1 µg / mL).

  About the cell which immunostained by the said method, Ki67 positive rate in a cardiac muscle cell was measured using In Cell Analyzer1000 (GE Healthcare). Ki67 nucleoprotein is expressed in proliferating cells in every cycle of the cell cycle (Scholzen & Gerdes, Journal of Cellular Physiology, 182: 311-22, 2000), thus detecting cells with an advanced cell cycle Used to do.

  The results are shown in FIG.

  From this result, miR-148a, miR-148b, miR-152 and miR-373 were identified as miRNAs having a proliferative action of cardiomyocyte prominently compared with the case where Negative control # 1 was used.

Example 3 Analysis of cell cycle progression markers by adenovirus-expressed miRNA forced expression in primary cultured cardiomyocytes
As an adenoviral vector for expressing miR-148a, miR-152, and miR-373, it was prepared using ViraPower ^™ Adenoviral Expression System (Invitrogen). That is, a human genomic DNA fragment containing the precursor sequence of each miRNA and several hundred adjacent nucleotides was amplified by PCR using the human genomic DNA as a template and the primers shown below.

hsa-miR-148a-F: 5'-caccgaacacacctgcaggaagaaact-3 '(SEQ ID NO: 11)
hsa-miR-148a-R: 5'-gttcccatttacagggtttaaccca-3 '(SEQ ID NO: 12)
hsa-miR-152-F: 5'-caccgtcccagactcggctcccatca-3 '(SEQ ID NO: 13)
hsa-miR-152-R: 5'-actcgaggtggacaccctgtgt-3 '(SEQ ID NO: 14)
hsa-miR-373-F: 5'-caccgtgaccaaggggctgtatgca-3 '(SEQ ID NO: 15)
hsa-miR-373-R: 5'-ctgcccaccccagaatatgcca-3 '(SEQ ID NO: 16).

  Insert these PCR products into the pENTR / D-TOPO vector (Invitrogen), confirm the nucleotide sequence, and recombine into the pAd / CMV / V5-DEST vector (Invitrogen) using the Gateway system (Invitrogen). PAd / CMV-miR-148a, pAd / CMV-miR-152, and pAd / CMV-miR-373 were prepared. Next, these vectors were cleaved with the restriction enzyme PacI, and transfected into human kidney-derived 293A cells using Lipofectamine 2000 (Invitrogen), so that recombinant adenovirus Ad-miR-148a, Ad-miR-152, And Ad-miR-373 were prepared. The recombinant adenovirus produced by the above method is constructed so that the precursor of each inserted miRNA is expressed under the control of the CMV promoter and can be highly expressed in mammalian cells. A precursor of miRNA expressed in a mammal becomes a functional mature miRNA by a group of enzymes in the mammalian cell.

  In this experiment, a commercially available recombinant adenovirus for expressing LacZ, Ad-LacZ (Takara Bio Inc.) was used.

Subsequently, a high titer virus solution was prepared for each recombinant virus, and the titer of the prepared virus solution was determined by plaque assay using 293A cells, all within the range of 10 ⁹ to 10 ¹⁰ pfu / mL. Met. In the present specification, the number of live virus particles to be infected per cell is hereinafter expressed as a multiplicity of infection (moi). That is, moi = 1 is represented when one cell is infected with one virus particle.

  Further, each prepared recombinant virus was added to cardiomyocytes at moi = 10 to 1000, and after 2 days, total RNA containing miRNA was collected using miRNeasy mini kit (Qiagen). Using 10 ng of the total RNA, cDNA was synthesized using the reverse transcription primers specific to each miRNA attached to TaqMan MicroRNA reverse transcription kit (Applied Biosystems) and TaqMan MicroRNA Assay (Applied Biosystems), Subsequently, using the forward and reverse primers specific for each miRNA included in the TaqMan MicroRNA Assay (Applied Biosystems) and TaqMan probe, real-time PCR is performed using the Applied Biosystems 7900HT Fast Real time PCR system. The expression level as miRNA was quantified.

  RNU6B was used as an endogenous control, and the expression level was normalized. As a result, it was confirmed that mature miRNA was expressed in a dose-dependent manner.

  As a negative control, recombinant viruses Ad-miR-148a, Ad-miR-152 and Ad-miR-373 prepared in this manner, Ad-LacZ as a negative control, cardiomyocytes (100,000 cells / well in a 24-well plate), moi = 10-100 and cells were fixed with 4% paraformaldehyde after 2-4 days.

Next, after anti-Ki67 antibody (ThermoSCIENTIFIC) (1: 200 dilution) and anti-TroponinT antibody (ThermoSCIENTIFIC) (1: 200 dilution) were reacted, Alexa Fluor ^™ labeled antibodies (Alexa-488 and Alexa-568; Molecular Probes) (both 1: 400 dilution). Cell nuclei were stained with DAPI solution (1 μg / mL).

  About the cell which immunostained by the said method, Ki67 positive rate in a cardiac muscle cell was measured using In Cell Analyzer1000.

  The results are shown in FIG. In this figure, None indicates unprocessed.

  From this result, in any of miR-148a, miR-152 and miR-373, compared with the negative control (LacZ), Ki67 which is a cell cycle progression marker becomes positive and the cell cycle of cardiomyocytes is increased. It was confirmed that it has an effect of promoting proliferation of cardiomyocytes.

Example 4 Analysis of cell division marker by miRNA forced expression or adenovirus-expressed miRNA forced expression in primary cultured cardiomyocytes In the same manner as in Example 2, miR-148a, miR-148b, miR-152, miR-373 and negative As a control, Negative control # 1 was transfected into cardiomyocytes. In addition, Ad-miR-148a, Ad-miR-152, Ad-miR-373 and negative control Ad-LacZ were added to cardiomyocytes in the same manner as in Example 3. Each cell was fixed with 4% paraformaldehyde 2-3 days after addition.

Next, after anti-phosphorylated histone H3 (H3P) antibody (MILLIPORE) (1: 200 dilution) and anti-TroponinT antibody (ThermoSCIENTIFIC) (1: 200 dilution) were reacted, Alexa Fluor ^TM labeled antibody (Alexa-488) And Alexa-568; Molecular Probes) (both diluted 1: 400). Cell nuclei were stained with DAPI solution (1 μg / mL).

  About the cell which immunostained by the said method, the H3P positive rate in a cardiac muscle cell was measured using In Cell Analyzer1000. Since histone H3 Ser-10 phosphorylation is closely related to the aggregation of chromatin observed during cell mitosis (Adamas, R, The Journal of Cell Biology, 153: p. 865-880, 2001) ), Used as a marker of cell division.

  The results are shown in Figs. 3-1 and 3-2. In this figure, None indicates unprocessed. Further, a micrograph of the cells subjected to the above immunostaining is shown in Fig. 3-3.

  From this result, in any of miR-148a, miR-152 and miR-373, in the case of Fig. 3-1, compared to the negative control (Negative control # 1), in the case of Fig. 3-2 Compared with the negative control (Ad-LacZ), the cell division marker H3P is positive, and cells that are undergoing mitosis and cytokinesis are observed, which increases cardiomyocyte cell division. These microRNAs were confirmed to have a cardiomyocyte proliferation promoting action.

Example 5: Examination of cardiomyocyte proliferation in vivo
In order to confirm whether the expression of miR-148a, miR-152, miR-373 causes proliferation of cardiomyocytes in vivo, the recombinant viruses prepared in Example 3 Ad-miR-148a, Ad-miR -152 and Ad-miR-373 (each 1 × 10 ⁹ pfu / mL), Wistar rats (250-300 g) are opened and visually 30G needles are placed in a total of 4 apical regions. A total volume of 200 μL was injected. As a negative control, miR-141 was used, and the target sequence was expressed as follows according to the method of Example 3, using human genomic DNA as a template:
hsa-miR-141-F: 5'-caccgcagggatcctgggcctga-3 '(SEQ ID NO: 17)
hsa-miR-141-R: 5'-cgggaagacaatggaggtgcct-3 '(SEQ ID NO: 18)
The PCR product was used as described in Example 3 to prepare Ad-miR-141, and Ad-miR-141 (1 × 10 ⁹ pfu / mL) was used as a Wistar rat. (250-300 g) was opened, and a total amount of 200 μL was injected into a total of four apical regions using a 30 G needle.

  Four days after the injection, 4% paraformaldehyde was perfused to fix the heart tissue, sections were prepared, anti-Ki67 antibody (ThermoSCIENTIFIC) (1: 500 dilution) and anti-TroponinT antibody (ThermoSCIENTIFIC) (1: (600 dilution), and then stained and visualized in green and red using Alexa Fluor ™ labeled antibodies (Alexa-488 and Alexa-568; Molecular Probes) (both 1: 400 dilution), respectively. Cell nuclei were stained and visualized in blue using DAPI.

  Stained heart tissue sections are observed using a fluorescence microscope, and DAPI and Troponin T co-positive cells are cardiomyocytes, Ki67 and Troponin T co-positive cells are cardiomyocytes with an advanced cell cycle, and the cell cycle progresses The percentage of cardiomyocytes in the blood was measured. The number of cells measured in each individual was 300 or more. The results are shown in FIG. In FIG. 4, it was found that the proportion of Ki67 positive cardiomyocytes was clearly increased in the heart infected with miR-148a, miR-152 or miR-373 adenovirus. On the other hand, Ki67 positive cardiomyocytes were hardly observed in the heart infected with the negative control miR-141 adenovirus.

  From this result, by expressing miR-148a, miR-152 and miR-373 in vivo, Ki67, a cell cycle progression marker, became positive, the cell cycle of cardiomyocytes was increased, and proliferative ability was acquired. It was shown that.

SEQ ID NO: 1: Seed sequence common to the 5 ′ side of the miR-148 family;
SEQ ID NO: 2: Seed sequence common to the 5 ′ side of the miR-373 family;
SEQ ID NO: 11: forward primer for amplifying the coding region of the miR-148a gene, hsa-miR-148a-F;
SEQ ID NO: 12: reverse primer for amplifying the coding region of the miR-148a gene, hsa-miR-148a-R;
SEQ ID NO: 13: forward primer for amplifying the coding region of the miR-152 gene, hsa-miR-152-F;
SEQ ID NO: 14: reverse primer for amplifying the coding region of the miR-152 gene, hsa-miR-152-R;
SEQ ID NO: 15: forward primer for amplifying the coding region of the miR-373 gene, hsa-miR-373-F;
SEQ ID NO: 16: reverse primer for amplifying the coding region of the miR-373 gene, hsa-miR-373-R;
SEQ ID NO: 17: forward primer for amplifying the coding region of miR-141 gene, hsa-miR-141-F;
SEQ ID NO: 18: reverse primer for amplifying the coding region of the miR-141 gene, hsa-miR-141-R;
See Table 1 for other sequences.

Claims (20)

  A pharmaceutical composition for treating heart disease, comprising a nucleic acid that defines miRNA having an effect of promoting proliferation of cardiomyocytes.   2. The pharmaceutical composition according to claim 1, wherein the miRNA is a substance selected from the group consisting of a mature miRNA and a precursor of the miRNA, and variants and similar substances thereof.   The pharmaceutical composition according to claim 1 or 2, wherein the miRNA is a substance comprising a seed sequence consisting of SEQ ID NO: 1 or SEQ ID NO: 2.   Substances comprising a seed sequence consisting of SEQ ID NO: 1 are miR-148a (SEQ ID NO: 3), miR-148b (SEQ ID NO: 5), miR-152 (SEQ ID NO: 7), and their 4. The pharmaceutical composition according to claim 3, which is a substance selected from the group consisting of precursors and their variants and similar substances.   4. The substance comprising a seed sequence consisting of SEQ ID NO: 2 is a substance selected from the group consisting of miR-373 (SEQ ID NO: 9) and its precursor, and its variants and similar substances. A pharmaceutical composition according to 1.   6. The pharmaceutical composition according to any one of claims 1 to 5, comprising a nucleic acid defining miRNA having a cardiomyocyte proliferation promoting action as an expression vector for introduction and expression in cardiomyocytes.   The pharmaceutical composition according to claim 6, wherein the vector is a viral vector.   The medicament according to any one of claims 1 to 7, wherein the miRNA is a miRNA derivative comprising at least one modified internucleoside linkage, at least one modified sugar moiety, at least one modified base, or a combination thereof. Composition.   The pharmaceutical composition according to any one of claims 1 to 8, wherein the heart disease is myocardial infarction, ischemic heart disease, congestive heart failure, hypertrophic cardiomyopathy, dilated cardiomyopathy, myocarditis or chronic heart failure.   A method for treating a heart disease, comprising introducing a nucleic acid defining miRNA having an effect of promoting proliferation of cardiomyocytes into a damaged site of an individual's heart to proliferate cardiomyocytes at the site.   Use of a nucleic acid that defines miRNA having a cardiomyocyte proliferation promoting action for the manufacture of a pharmaceutical composition for treating heart disease.   A nucleic acid that defines miRNA having a cardiomyocyte proliferation promoting action for use in the treatment of heart disease.   A method for proliferating cardiomyocytes, characterized in that a nucleic acid defining miRNA having a cardiomyocyte proliferation promoting action is introduced and expressed in cardiomyocytes. 14. The method according to claim 13, wherein the miRNA is a substance selected from the group consisting of a mature miRNA and a precursor of the miRNA, and variants and similar substances thereof.   15. The method according to claim 13 or 14, wherein the miRNA is a substance comprising a seed sequence consisting of SEQ ID NO: 1 or SEQ ID NO: 2.   Substances comprising a seed sequence consisting of SEQ ID NO: 1 are miR-148a (SEQ ID NO: 3), miR-148b (SEQ ID NO: 5), miR-152 (SEQ ID NO: 7), and their 16. The method of claim 15, which is a substance selected from the group consisting of precursors, and variants and analogs thereof.   16. The substance comprising a seed sequence consisting of SEQ ID NO: 2 is a substance selected from the group consisting of miR-373 (SEQ ID NO: 9) and its precursor, and variants and similar substances thereof. The method described in 1.   18. The method according to any one of claims 13 to 17, comprising introducing a nucleic acid defining miRNA having an effect of promoting proliferation of cardiomyocytes into the cardiomyocytes using an expression vector.   19. A method according to claim 18, wherein the vector is a viral vector.   The method according to any one of claims 13 to 19, wherein the miRNA is a miRNA derivative comprising at least one modified internucleoside linkage, at least one modified sugar moiety, at least one modified base, or a combination thereof. .